Energy-dependent AAA+ proteases have been implicated in controlling numerous physiologically significant pathways. The Lon AAA+ protease, which is conserved from bacteria to humans, has emerged as key contributor to the capacity of the cell to adapt to its ever-changing growth environment. Lon protease play a fundamental role in maintaining the ideal concentration of key regulatory proteins, and in re-sculpting the proteome by removing unfolded, aberrant, or damaged proteins in response to internal and external signals. Knowing how this versatile AAA+ protease and its cofactor(s) select target substrates for degradation is crucial to our understanding of its biological functions. Recent studies have provided compelling evidence to demonstrate that the Lon AAA+ proteases play critical roles in the regulation of virulence gene expression in a number of pathogenic bacteria. The long-term goal of this proposal is to establish the role of the AAA+ Lon proteases in the pathogenesis of bacteria that are required to adapt to environmental changes during their transmission from invertebrate vectors to mammalian hosts. The two specific aims of the proposal are designed to guide us in studies of two vector borne pathogens, Yersinia pestis, transmitted by fleas, and Borrelia burgdorferi, transmitted by ticks. Our aims are motivated by the hypothesis that ATP-fueled Lon proteases facilitate important physiological transitions by selectively removing key regulatory proteins surplus unwanted substrates. Further, we hypothesize that Lon protease selectivity is mediated by specificity enhancing factors (adaptor proteins) or selective expression of unique Lon orthologs with distinct substrate specificities. In Aim I, we will focus on the role and regulation of Lon protease in Yersinia pestis, the causative agent of plague, during the key physiological transition resulting in expression of the type III secretion system. We will focus particularly on the discovery and characterization of a novel and much sought after specificity-enhancing factor for Lon protease. We will investigate the mechanism by which this novel adaptor protein regulates substrate selection and degradation by Lon protease. In Aim II, we will focus on the role and regulation of Lon proteases in Borrelia burgdorferi, the etiological agent of Lyme diseases, with special focus on the unusual occurrence of two Lon proteases (Bb-Lon-1 and Bb-Lon-2), of which the former is expressed in blood. We will examine the merits of the hypothesis that the Bb-Lon-1 has a distinct substrate range and specificity, uniquely different from the canonical Lon proteases of other bacteria, including the Bb-Lon-2 protease. We will determine the substrate range and specificity of Borrelia Lon proteases and elucidate how Bb-Lon-1 remodels the spirochetal proteome and maintains ideal concentration of key regulatory proteins to facilitate the physiological transition triggered by exposure to mammalian blood. A detailed understanding of the substrate range and specificity of Lon proteases, and how remodeling of the bacterial proteome by Lon contributes to the fitness, survival, and virulence of bacteria will provide significant new insight into the principles of general and selective proteolysis.